Crohn's disease: loss of tolerance or a disorder of autophagy?

Crohn's disease (CD) is characterized by a breakdown of the intestinal epithelial barrier function leading to an uncontrolled immune response to bacterial antigens. Available data demonstrate that appropriate response and early host defense against invading bacteria are crucial to maintain tolerance towards commensal bacteria. When the mechanisms of early removal of invading bacteria are disturbed, a loss of tolerance and a full-blown adaptive immune reaction, which is mounted against the usually harmless commensal flora, are induced. Dysfunction of autophagy caused by genetic variations within CD susceptibility genes, such as ATG16L1 and IRGM, results in defective handling of intracellular and invading bacteria and causes prolonged survival and defective clearance of those microbes. Dysfunction of ATG16L1 and IRGM has also been shown to cause aberrant Paneth cell function and uncontrolled secretion of proinflammatory cytokines finally resulting in increased susceptibility to bacterial infection and the onset of colitis. Interestingly, autophagy can also be regulated by other CD susceptibility genes, such as NOD2 (nucleotide oligomerization domain 2) or PTPN2 (protein tyrosine phosphatase nonreceptor type 2) and the presence of the CD-associated variations within these genes results in similar effects. Taken together, more and more evidence suggests a close functional correlation between loss of tolerance and defective autophagy in CD patients. Therefore, most likely, the onset of CD is triggered by both a loss of tolerance as well as a dysfunction of autophagy, which finally results in the onset of chronic intestinal inflammation

Temperature-triggered in situ forming lipid mesophase gel for local treatment of ulcerative colitis

Ulcerative colitis is a chronic inflammatory bowel disease that strongly affects patient quality of life. Side effects of current therapies necessitate new treatment strategies that maximise the drug concentration at the site of inflammation, while minimizing systemic exposure. Capitalizing on the biocompatible and biodegradable structure of lipid mesophases, we present a temperature-triggered in situ forming lipid gel for topical treatment of colitis. We show that the gel is versatile and can host and release drugs of different polarities, including tofacitinib and tacrolimus, in a sustained manner. Further, we demonstrate its adherence to the colonic wall for at least 6 h, thus preventing leakage and improving drug bioavailability. Importantly, we find that loading known colitis treatment drugs into the temperature-triggered gel improves animal health in two mouse models of acute colitis. Overall, our temperature-triggered gel may prove beneficial in ameliorating colitis and decreasing adverse effects associated with systemic application of immunosuppressive treatments

Stepwise Development of an in vitro Continuous Fermentation Model for the Murine Caecal Microbiota

Murine models are valuable tools to study the role of gut microbiota in health or disease. However, murine and human microbiota differ in species composition, so further investigation of the murine gut microbiota is important to gain a better mechanistic understanding. Continuous in vitro fermentation models are powerful tools to investigate microbe-microbe interactions while circumventing animal testing and host confounding factors, but are lacking for murine gut microbiota. We therefore developed a novel continuous fermentation model based on the PolyFermS platform adapted to the murine caecum and inoculated with immobilized caecal microbiota. We followed a stepwise model development approach by adjusting parameters [pH, retention time (RT), growth medium] to reach fermentation metabolite profiles and marker bacterial levels similar to the inoculum. The final model had a stable and inoculum-alike fermentation profile during continuous operation. A lower pH during startup and continuous operation stimulated bacterial fermentation (115 mM short-chain fatty acids at pH 7 to 159 mM at pH 6.5). Adjustments to nutritive medium, a decreased pH and increased RT helped control the in vitro Enterobacteriaceae levels, which often bloom in fermentation models, to 6.6 log gene copies/mL in final model. In parallel, the Lactobacillus, Lachnospiraceae, and Ruminococcaceae levels were better maintained in vitro with concentrations of 8.5 log gene copies/mL, 8.8 log gene copies/mL and 7.5 log gene copies/mL, respectively, in the final model. An independent repetition with final model parameters showed reproducible results in maintaining the inoculum fermentation metabolite profile and its marker bacterial levels. Microbiota community analysis of the final model showed a decreased bacterial diversity and compositional differences compared to caecal inoculum microbiota. Most of the caecal bacterial families were represented in vitro, but taxa of the Muribaculaceae family were not maintained. Functional metagenomics prediction showed conserved metabolic and functional KEGG pathways between in vitro and caecal inoculum microbiota. To conclude, we showed that a rational and stepwise approach allowed us to model in vitro the murine caecal microbiota and functions. Our model is a first step to develop murine microbiota model systems and offers the potential to study microbiota functionality and structure ex vivo

Spermidine ameliorates colitis via induction of anti-inflammatory macrophages and prevention of intestinal dysbiosis

BACKGROUND AND AIMS: Exacerbated immune activation, intestinal dysbiosis, and a disrupted intestinal barrier are common features among inflammatory bowel disease (IBD) patients. The polyamine spermidine, which is naturally present in all living organisms, is an integral component of the human diet, and exerts beneficial effects in human diseases. Here, we investigated whether spermidine treatment ameliorates intestinal inflammation and offers therapeutic potential for IBD treatment. METHODS: We assessed the effect of oral spermidine administration on colitis severity in the T cell transfer colitis model in Rag2 -/- mice by analysis of endoscopy, histology, and molecular inflammation markers. The effects on the intestinal microbiome were determined by 16S sequencing of mouse feces. The impact on intestinal barrier integrity was evaluated in co-cultures of patient-derived macrophages with intestinal epithelial cells. RESULTS: Spermidine administration protected mice from intestinal inflammation in a dose-dependent manner. While T helper cell subsets remained unaffected, spermidine promoted anti-inflammatory macrophages and prevented the microbiome shift from Firmicutes and Bacteroides to Proteobacteria, maintaining a healthy gut microbiome. Consistent with spermidine as a potent activator of the anti-inflammatory molecule protein tyrosine phosphatase non-receptor type 2 (PTPN2), its colitis-protective effect was dependent on PTPN2 in intestinal epithelial cells and in myeloid cells. The loss of PTPN2 in epithelial and myeloid cells, but not in T cells, abrogated the barrier-protective, anti-inflammatory effect of spermidine and prevented the anti-inflammatory polarization of macrophages. CONCLUSION: Spermidine reduces intestinal inflammation by promoting anti-inflammatory macrophages, maintaining a healthy microbiome, and preserving epithelial barrier integrity in a PTPN2-dependent manner

Glycoprotein (GP)96 is essential for maintaining intestinal epithelial architecture by supporting its self-renewal capacity

BACKGROUND & AIMS Glycoprotein (GP)96 is an endoplasmic reticulum (ER)-resident master chaperone for cell surface receptors including the Wnt co-receptors LRP5/6. Intestinal epithelial cells (IEC)-specific deletion of Gp96 is embryonically lethal. However, the role of GP96 in adult intestinal tissue and especially within the intestinal stem cell (ISC) niche has not been studied so far. Here, we investigated how GP96-loss interferes with intestinal homeostasis by compromising viability, proliferation and differentiation of IEC. METHODS Tamoxifen was used to induce Cre-mediated deletion of Gp96 in GP96-Villin$^{creERT2}$ mice and intestinal organoids. With H&E- and immunofluorescence staining we assessed alterations in intestinal morphology and the presence and localization of IEC-types. Real-time PCR and Western blot analysis were performed to explore the molecular mechanisms underlying the severe phenotype of Gp96 KO mice and organoids. RESULTS IEC-specific deletion of Gp96 in adult mice resulted in a rapid degeneration of the stem cell niche, followed by a complete eradication of the epithelial layer and death within few days. These effects were due to severe defects in ISC renewal and premature ISC differentiation, which resulted from defective Wnt and Notch signaling. Furthermore, depletion of GP96 led to massive induction of ER stress. While effects on ISC renewal and adequate differentiation were partly reversed upon activation of Wnt/Notch signaling, viability could not be restored, indicating that reduced viability was mediated by other mechanisms. CONCLUSIONS Our work demonstrates that GP96 plays a fundamental role in regulating ISC fate and epithelial regeneration and is therefore indispensable for maintaining intestinal epithelial homeostasis

The presence of genetic risk variants within PTPN2 and PTPN22 is associated with intestinal microbiota alterations in Swiss IBD cohort patients.

BACKGROUND Genetic risk factors, intestinal microbiota and a dysregulated immune system contribute to the pathogenesis of inflammatory bowel disease (IBD). We have previously demonstrated that dysfunction of protein tyrosine phosphatase non-receptor type 2 (PTPN2) and PTPN22 contributes to alterations of intestinal microbiota and the onset of chronic intestinal inflammation in vivo. Here, we investigated the influence of PTPN2 and PTPN22 gene variants on intestinal microbiota composition in IBD patients. METHODS Bacterial DNA from mucosa-associated samples of 75 CD and 57 UC patients were sequenced using 16S rRNA sequencing approach. Microbial analysis, including alpha diversity, beta diversity and taxonomical analysis by comparing to PTPN2 (rs1893217) and PTPN22 (rs2476601) genotypes was performed in QIIME, the phyloseq R package and MaAsLin pipeline. RESULTS In PTPN2 variant UC patients, we detected an increase in relative abundance of unassigned genera from Clostridiales and Lachnospiraceae families and reduction of Roseburia when compared to PTPN2 wild-type (WT) patients. Ruminoccocus was increased in PTPN22 variant UC patients. In CD patients with severe disease course, Faecalibacterium, Bilophila, Coprococcus, unclassified Erysipelotrichaeceae, unassigned genera from Clostridiales and Ruminococcaceae families were reduced and Bacteroides were increased in PTPN2 WT carriers, while Faecalibacterium, Bilophila, Coprococcus, and Erysipelotrichaeceae were reduced in PTPN22 WT patients when compared to patients with mild disease. In UC patients with severe disease, relative abundance of Lachnobacterium was reduced in PTPN2 and PTPN22 WT patients, Dorea was increased in samples from PTPN22 WT carriers and an unassigned genus from Ruminococcaceae gen. was increased in patients with PTPN2 variant genotype. CONCLUSIONS We identified that IBD-associated genetic risk variants, disease severity and the interaction of these factors are related to significant alterations in intestinal microbiota composition of IBD patients

Titanium dioxide nanoparticles exacerbate DSS-induced colitis: role of the NLRP3 inflammasome

OBJECTIVE: Western lifestyle and diet are major environmental factors playing a role in the development of IBD. Titanium dioxide (TiO2) nanoparticles are widely used as food additives or in pharmaceutical formulations and are consumed by millions of people on a daily basis. We investigated the effects of TiO2 in the development of colitis and the role of the nucleotide-binding oligomerisation domain receptor, pyrin domain containing (NLRP)3 inflammasome. DESIGN: Wild-type and NLRP3-deficient mice with dextran sodium sulfate-induced colitis were orally administered with TiO2 nanoparticles. The proinflammatory effects of TiO2 particles in cultured human intestinal epithelial cells (IECs) and macrophages were also studied, as well as the ability of TiO2 crystals to traverse IEC monolayers and accumulate in the blood of patients with IBD using inductively coupled plasma mass spectrometry. RESULTS: Oral administration of TiO2 nanoparticles worsened acute colitis through a mechanism involving the NLRP3 inflammasome. Importantly, crystals were found to accumulate in spleen of TiO2-administered mice. In vitro, TiO2 particles were taken up by IECs and macrophages and triggered NLRP3-ASC-caspase-1 assembly, caspase-1 cleavage and the release of NLRP3-associated interleukin (IL)-1β and IL-18. TiO2 also induced reactive oxygen species generation and increased epithelial permeability in IEC monolayers. Increased levels of titanium were found in blood of patients with UC having active disease. CONCLUSION: These findings indicate that individuals with a defective intestinal barrier function and pre-existing inflammatory condition, such as IBD, might be negatively impacted by the use of TiO2 nanoparticles

Protein tyrosine phosphatase non-receptor type 2 controls colorectal cancer development

Protein tyrosine phosphatase non-receptor type 2 (PTPN2) recently emerged as a promising cancer immunotherapy target. We set to investigate the functional role of PTPN2 in the pathogenesis of human colorectal carcinoma (CRC) as its role in immune-silent solid tumors is poorly understood. We demonstrate that in human CRC, increased PTPN2 expression and activity correlated with disease progression and decreased immune responses in tumor tissues. Particularly, stage II and III tumors displayed enhanced PTPN2 protein expression in tumor-infiltrating T-cells and increased PTPN2 levels negatively correlated with PD1, CTLA4, STAT1 and granzyme A. In vivo, T-cell and dendritic cell-specific PTPN2 deletion reduced tumor burden in several CRC models by promoting CD44+ effector/memory T-cells, as well as CD8+ T-cell infiltration and cytotoxicity into the tumor. In direct relevance to CRC treatment, T-cell-specific PTPN2 deletion potentiated anti-PD-1 efficacy and induced anti-tumor memory formation upon tumor re-challenge in vivo. Our data suggest a role for PTPN2 in suppressing anti-tumor immunity and promoting tumor development in CRC patients. Our in vivo results uncover PTPN2 as a key player in controlling immunogenicity of CRC, with the strong potential to be exploited to promote cancer immunotherapy

Elevated oxysterol levels in human and mouse livers reflect nonalcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH), a primary cause of liver disease, leads to complications such as fibrosis, cirrhosis, and carcinoma, but the pathophysiology of NASH is incompletely understood. Epstein Barr virus induced G protein coupled receptor 2 (EBI2) and its oxysterol ligand 7α,25-dihydroxycholesterol (7α,25-diHC) are recently discovered immune regulators. Several lines of evidence suggest a role of oxysterols in NASH pathogenesis, but rigorous testing has not been performed. We measured oxysterol levels in livers of NASH patients by liquid chromatography-mass spectrometry and tested the role of the EBI2-7α,25-diHC-system in a murine feeding model of NASH. Free oxysterol profiling in livers from NASH patients revealed a pronounced increase in 24- and 7-hydroxylated oxysterols in NASH compared to controls. Levels of 24- and 7-hydroxylated oxysterols correlated with histological NASH activity. Histological analysis of murine liver samples demonstrated ballooning and liver inflammation. No significant genotype related differences were observed in Ebi2-/- animals and animals with defects in the 7α,25-diHC synthesizing enzymes CH25H and CYP7B1 compared to wildtype littermate controls,arguing against an essential role of these genes in NASH pathogenesis. Elevated 24- and 7-hydroxylated oxysterol levels were confirmed in murine NASH liver samples. Our results suggest increased bile acid synthesis in NASH samples, as judged by enhanced level of 7α- hydroxycholest-4-en-3-one, and impaired 24S-hydroxycholesterol metabolism as characteristic biochemical changes in livers affected by NASH

The role for protein tyrosine phosphatase non-receptor type 22 in regulating intestinal homeostasis

Inflammatory bowel disease represents a chronic intestinal inflammation. Recent knowledge suggests a crucial role for genetic, immunological and bacterial factors in inflammatory bowel disease pathogenesis. Variations within the gene locus encoding PTPN22 have been associated with inflammatory bowel disease. PTPN22 is critically involved in controlling immune cell activation and thereby plays an important role in maintaining intestinal homeostasis. Although in B and T cells the mechanism showing how PTPN22 affects cell signalling pathways is well studied, its role in myeloid cells remains less defined. Regulation of the innate immune system plays an essential role in the intestine, and levels of PTPN22 in myeloid cells are drastically reduced in the intestine of inflammatory bowel disease patients. Therefore, additional studies to define the role of PTPN22 in myeloid cells might clearly enhance our understanding of how PTPN22 contributes to intestinal homeostasis